The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection time scales for single molecules in solution without immobilizing them onto a substrate surface and applying external fields are much sought-after. Here, we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-time scale analysis of nanoscopic material building blocks such as single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nanotrapping geometries down to 100 nm in height. We provide a rigorous description and characterization of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nanotrapping and nanochannel geometries within microchannel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nanotrap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a 3- to 5-fold increase in average residence time in the detection volume of nanotraps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby microchannel. Our approach thus opens up the possibility for single-molecule studies at prolonged observational-time scales to analyze and detect functionalized nanoparticles and protein assemblies in solution without the need for surface immobilization.

We design and fabricate a grating coupler for interfacing suspended silicon photonic membranes with free-space optics while being compatible with single-step lithography and etching in 220 nm silicon device layers. The grating coupler design simultaneously and explicitly targets both high transmission into a silicon waveguide and low reflection back into the waveguide by means of a combination of a two-dimensional shape-optimization step followed by a three-dimensional parameterized extrusion. The designed coupler has a transmission of −6.6 dB (21.8 %), a 3 dB bandwidth of 75 nm, and a reflection of −27 dB (0.2 %). We experimentally validate the design by fabricating and optically characterizing a set of devices that allow the subtraction of all other sources of transmission losses as well as the inference of back-reflections from Fabry-Pérot fringes, and we measure a transmission of 19 % ± 2 %, a bandwidth of 65 nm and a reflection of 1.0 % ± 0.8 %.

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle’s size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics.